Human cytomegalovirus pp65 peptide-induced autoantibodies cross-reacts with TAF9 protein and induces lupus-like autoimmunity in BALB/c mice

Abstract

Human cytomegalovirus (HCMV) has been linked to the triggering of systemic lupus erythematosus (SLE). We proposed that B cell epitope region of HCMV phosphoprotein 65 (HCMVpp65)_422-439 mimics an endogenous antigen and initiates lupus-like autoimmunity. Amino acid homology between HCMVpp65_422-439 and TAF9_134-144 (TATA-box binding protein associated factor 9, TAF9) was investigated using a similarity search in NCBI protein BLAST program (BLASTP). A murine model was used to confirm their antigenicity and ability to induce lupus-like symptoms. HCMVpp65_422-439 induced immune responses with the presence of specific antibodies against HCMVpp65_422-439 and TAF9_134-144, as well as anti-nuclear and anti-double-stranded (ds)DNA antibodies that are characteristic of SLE. In addition, the majority of HCMVpp65_422-439 and TAF9_134-144 immunized mice developed proteinuria, and their renal pathology revealed glomerulonephritis with typical abnormalities, such as mesangial hypercellularity and immune complex deposition. Immunoglobulin eluted from the glomeruli of HCMVpp65_422-439 immunized mice showed cross-reactivity with TAF9_134-144 and dsDNA. Increased anti-TAF9 antibody activity was also observed in the sera from SLE patients compared with healthy people and disease controls. Molecular mimicry between HCMVpp65 peptide and host protein has the potential to drive lupus-like autoimmunity. This proof-of-concept study highlights the mechanisms underlying the link between HCMV infection and the induction of SLE.

Figure 1

Association between anti-HCMVpp65_422-439 and anti-TAF9 activities in SLE. (a) Alignment of the amino acid sequences for HCMVpp65_422-439, HCMVpp65_425-436, HCMVpp65_428-437, HCMVpp65_430-439 and TAF9_134-144. HCMVpp65_425-436 and HCMVpp65_428-437 B cell epitopes were recognized by purified anti-HCMVpp65_422-439 IgG from the sera of human SLE, and HCMVpp65_422-439 immunized mice, respectively. The TAF9 fragment contains a TAF9_136-142 epitope like HCMVpp65_428-437 (b,c) ELISA analysis for the detection of IgG against HCMVpp65_422-439 and TAF9 protein using sera from patients with SLE (n = 67), SS (n = 23), RA (n = 38), AS (n = 18), gout (n = 40) and healthy controls (n = 72). (d) Western blotting for the examination of IgG antibodies response to HCMVpp65 and TAF9 proteins. There was a positive reaction to HCMVpp65 and TAF9 proteins using dual positive sera in the ELISA test. The positivity was defined as the mean + 3SEM of the sera. Sera with an optical density (O.D.)₄₅₀ > 0.932 of anti-TAF9 activity and O.D.₄₅₀ > 0.835 of anti-HCMVpp65_422-439 activity were considered to be dual positive sera. Asterisks indicate the sera are positive for HCMVpp65 or TAF9. SLE: sera of SLE patients; N and R: serum of normal population and patient with RA, respectively. P: positive control; His-tagged HCMVpp65 or TAF9 protein was recognized by the HRP-conjugated anti-His-tag antibody. Uncropped blots related to Fig. 1 were shown in Supplementary Fig. S4. (e) Correlation of anti-HCMVpp65_422-439 and anti-TAF9 activities in patients with rheumatic diseases and healthy controls. Data are shown as the mean ± SEM of three independent experiments.

Figure 2

Detection of cross-reactive IgG antibodies against immunized peptides and cellular proteins and dsDNA in the sera of HCMVpp65_422-439, TAF9_134-144, and SA-C3d immunized mice. (a) Schematic diagram of the peptide-C3d complex. (b) Diagram of the immunization schedule. (c–f) ELISA analysis for anti-HCMVpp65_422-439, anti-TAF9_134-144, anti-HeLa lysate and anti-dsDNA activity from the sera of HCMVpp65_422-439 (n = 10), TAF9_134-144 (n = 10) and SA-C3d (n = 5) immunized mice. 250x diluted sera and 1 μg/well HCMVpp65_422-439 peptide or TAF9 protein were used. (g) ELISA analysis for IgG subclasses of anti-dsDNA antibodies from HCMVpp65_422-439 (n = 8) or TAF9_134-144 (n = 7) immunized mice at 12 weeks after immunization at 1:80 dilution. Data are shown as the mean ± SEM of three independent experiments.

Figure 3

ELISA analysis for HCMVpp65_422-439, TAF9_134-144, and TAF9-specific IgG purified from pooled sera of immunized mice at 10-12 weeks after immunization or patients with SLE. ELISA analysis for anti-HCMVpp65_422-439, anti-TAF9_134-144, anti-TAF9, and anti-dsDNA activities using purified IgG from (a) immunized mice sera and (b) human SLE sera. A total of 1 μg anti-HCMVpp65_422-439 or anti-TAF9_134-144 IgG antibody, or 100 μl eluted anti-TAF9 IgG fraction (1 ml/tube) was used. ELISA competitive analysis for anti-HCMVpp65_422-439, anti-TAF9_134-144, and anti-TAF9 activities using purified IgG from the sera of (c–e) immunized mice and (f–h) human SLE sera. For the competitive assay, 2 μg/well HCMVpp65_422-439, TAF9_134-144, dsDNA or TAF9 protein was used as competitor agents. Data are shown as the mean ± SEM of three independent experiments.

Figure 4

Investigating IgG deposition and the histopathology of glomeruli from HCMVpp65_422-439, TAF9_134-144, and SA-C3d immunized mice at 16 weeks after immunization. (a) Kidney sections from HCMVpp65_422-439 and TAF9_134-144 immunized mice were stained with FITC-conjugated anti-mouse (a1) IgG, (a2) IgM and (a3) C3. Immunoglobulin deposition was not found in glomeruli from (a4) SA-C3d immunized mice. Scale bar represents 100 nm (b) Kidney sections from HCMVpp65_422-439, TAF9_134-144, and SA-C3d immunized mice at 16 weeks post-immunization were stained with FITC-conjugated anti-mouse (b1) IgG1, (b2) IgG2a (b3) IgG2b and (b4) IgG3. Scale bar represents 100 nm. (c) Hematoxylin and eosin staining of the glomerular from (c1) SA-C3d (c2) TAF9_134-144 and (c3) HCMVpp65_422-439 immunized mice. Scale bar represents 35 nm. (d) Glomerular tuft area. Glomerular hypertrophy is evaluated in three groups of mice, (e) Mesangial matrix expansion index. The ratio of expanded mesangial surface area to the total glomerular surface area. (f) The glomerulonephritis score of renal lesions from immunized mice. (g) Glomerulonephritis lesion counts. One hundred glomeruli per mouse were counted without overlapping. Ear holes produced by an ear punch device are used to identify individual mouse. L: left ear; R: right ear. (g) Mice developed proteinuria at 4, 8 12, 14, and 16 weeks after immunization. Data are shown as the mean ± SEM of three independent experiments. The full images related to Fig. 4 were shown in Supplementary Fig. S4.

Figure 5

Identification of IgG antibody deposition in the glomeruli of immunized mice. (a) Western blot analysis of IgG eluted from the glomeruli of HCMVpp65_422-439 and TAF9_134-144 immunized mice against full-length HCMVpp65 or TAF9 protein. (b) ELISA analysis for anti-HCMVpp65_422-439, anti-TAF9_134-144, anti-TAF9, and anti-dsDNA activities using mouse IgG purified from glomerular deposition. 1 μg of eluted anti-HCMVpp65_422-439 or TAF9_134-144 IgG antibody was used for the test. (c) Representative images of Crithidia luciliae staining with 0.5 μg eluted IgG. Full images/blots were shown in Supplementary Fig. S4 (d) Isotypes of eluted IgG against dsDNA. Flow-through was used as the negative control. (e–f) ELISA competitive analysis for anti-HCMVpp65_422-439 and anti-TAF9_134-144 antibody activities using eluted IgG from the glomeruli of immunized mice. For the competitive assay, 2 μg/well of HCMVpp65_422-439, TAF9_134-144, dsDNA, or TAF9 protein was used as a competitor. (g) Mapping of critical amino acid residues on TAF9_136-142 peptide. 1 μg of amino acid substituted synthetic peptides, and 1 μg eluted IgG antibodies from glomeruli of HCMVpp65_422-439, or TAF9_134-144 immunized mice, and IgG purified from SLE sera was used for the test. Data are shown as the mean ± SEM of three independent experiments.